CN105247067A - fusion protease - Google Patents

Abstract

This invention relates to novel bifunctional fusion proteases useful for manufacturing a mature protein from a fusion protein. More specifically the present invention relates to bifunctional fusion proteases comprising a picornaviral 3C protease and a Xaa-Pro-dipeptidyl aminopeptidase.

Description

fusion protease

technical field

The present invention relates to the technical field of protein expression and protein chemistry, wherein mature proteins are released from fusion proteins.

Background technique

Recombinant protein technology enables the production of large quantities of a desired protein available for its biological activity. Such proteins are usually expressed as recombinant fusion proteins in microbial host cells. Mature proteins (proteins of interest) are often linked to fusion partners or smaller amino acid extensions to increase expression levels, improve solubility, facilitate protein folding, or facilitate purification and downstream processing.

Removal of the fusion partner protein from the fusion protein to release the mature protein with native N- and C-termini may be critical for maintaining the intact biological activity of the protein as well as for drug regulation purposes.

Currently, the few proteases available for the removal of fusion partners from fusion proteins are available as economically sustainable enzymes for industrial use, leaving the native N-terminus in the released mature target protein.

One such enzyme is enterokinase, however, its lack of specificity prevents widespread use. Other such enzymes are Factor Xa, trypsin, clostripain, thrombin, TEV or rhinovirus 3C protease, all of which either lack specificity since most proteins contain an internal secondary An amino acid extension is left at the C- or N-terminus.

Waugh, Protein Expr. Purif. 80:283-293 (2011) discloses a review of enzymatic reagents for the removal of affinity tags.

WO92/10576 discloses the use of fusion proteins with DPP IV cleavable extended peptide moieties in pharmaceutical formulations.

Xin, Protein Expr. Purif. 2002, 24, pp530-538 discloses X-prolyl dipeptidyl aminopeptides from Lactococcus lactis for removal of N-terminal Pro-Pro from recombinant proteins Cloning, expression and application of the enzyme in Escherichia coli .

Bülow, TIBTECH 9:226-231 (1991) discloses a method for producing bifunctional enzymes by gene fusion.

Seo, Appl. Environ. Microbiol. 2000, 66, pp2484-2490 discloses a bifunctional fusion enzyme of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase.

In the pharmaceutical industry, protein drugs currently constitute a substantial portion of the competitive market and, therefore, efficient methods of manufacturing these protein drugs on a large scale are required. A key issue for the industrial use of fusion proteins remains the removal of the fusion protein partner from the fusion protein to release the intact mature protein.

Therefore, there is a need for an industrial method that specifically removes fusion partner proteins with an internal cleavage site in the immature protein and does not leave any amino acid stretches on the mature protein. Preferably, the removal of the fusion partner is performed using only a single enzyme that is readily prepared in an industrial process. There is also a need for methods that can do this for a wide variety of proteins under mild processing conditions to prevent unintended chemical and physical changes to mature proteins.

overview

It is an object of the present invention to provide a simple one-step method for providing mature proteins from fusion proteins.

Both picornavirus 3C protease and Xaa-Pro-dipeptidyl aminopeptidase (XaaProDAP) are very specific enzymes that exhibit complementary activities that have been found to be useful in the manufacture of protein pharmaceuticals. However, as proteolytic enzymes, they also cause problems with self-cleavage when fused into a bifunctional fusion protease.

Combining two enzymes in a fusion protease may have the advantage of favorable reaction kinetics due to the physical proximity of the two enzymes and thus fewer side reactions. Combining two enzymes in a fusion protease also has the advantage that only one reagent needs to be provided and used. Due to their larger size, the fusion protease can also be easily removed from the mature protein by a simple gel filtration method.

According to the first aspect of the present invention, there is provided a bifunctional fusion protease comprising picornavirus 3C protease and the catalytic domain of XaaProDAP. In one embodiment, the bifunctional fusion protease comprises picornavirus 3C protease and XaaProDAP.

According to a second aspect of the present invention, there is provided a bifunctional fusion protease comprising a protein of the following formula:

X-Y-Z (I) or Z-Y-X (II)

in

X is a picornavirus 3C protease or a functional variant thereof;

Y is an optional linker;

Z is Xaa-Pro-dipeptidyl aminopeptidase (XaaProDAP) or a functional variant thereof;

Wherein said fusion protease has substantially no self-cleavage activity capable of impairing at least one of the two proteolytic activities.

In one embodiment, the bifunctional fusion protease of the present invention has formula (I), that is, the picornavirus 3C protease or its functional variant is located at the N-terminal part of the bifunctional fusion protease.

In another embodiment, X is human rhinovirus type 14 3C protease (HRV14 3C) or a functional variant thereof.

In another embodiment, Z is an E.C. 3.4.14.11 enzyme or a functional variant thereof.

According to a third aspect of the present invention, there is provided a method for preparing the bifunctional fusion protease of the present invention, which comprises recombinantly expressing a protein comprising the bifunctional fusion protease in a host cell and subsequently isolating the bifunctional fusion protease.

In one embodiment, the method for preparing a bifunctional fusion protease includes Escherichia coli as the host cell.

According to a fourth aspect of the present invention there is provided the use of the bifunctional fusion protease of the present invention for removing N-terminal peptides or proteins from larger peptides or proteins.

Brief description of the drawings

Figure 1 shows the reducing SDS-PAGE of the purified bifunctional HRV14-XaaProDAP fusion protease (Protease 20986). Lane 1: protein markers. Numbers indicate sizes in kDa. Lane 2: Purified protease 20986.

Figure 2 shows the deconvoluted mass spectrum of RL27_EVLFQGP_PYY(3-36) after incubation with protease 20986 at 37°C for 3 hours using a 1:20 molar enzyme to substrate ratio (reaction 1). X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

Figure 3 shows the deconvoluted mass spectrum of RL27_EVLFQGP_PYY(3-36) after incubation with protease 20986 at 37°C for 3 hours using a 1:40 molar enzyme to substrate ratio (reaction 2). X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

Figure 4 shows the deconvoluted mass spectrum of RL27_EVLFQGP_PYY(3-36) after incubation with RL9-HRV14 3C protease for 3 hours at 37°C using a 1:20 molar enzyme to substrate ratio (reaction 3). X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

Figure 5 shows the deconvoluted mass spectrum of RL27_EVLFQGP_PYY(3-36) after incubation with RL9-HRV14 3C protease for 3 hours at 37°C using a 1:40 molar enzyme to substrate ratio (reaction 4). X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

Figure 6 shows the deconvoluted mass spectrum of RL27_EVLFQGP_glucagon after overnight incubation at 4°C with protease 20986 using a 1 :500 molar enzyme to substrate ratio (reaction 12). X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

Figure 7 shows the deconvoluted mass spectrum of RL27_EVLFQGP_glucagon after overnight incubation at 4°C with protease 28994 using a 1:100 molar enzyme to substrate ratio (reaction 13). X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

Figure 8 shows the deconvoluted mass spectrum of RL27_EVLFQGP_glucagon after overnight incubation at 4°C with protease 28996 using a 1 :500 molar enzyme to substrate ratio (reaction 16). X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

Figure 9 shows the deconvoluted mass spectrum of RL27_EVLFQGP_glucagon after overnight incubation at 4°C with protease 28997 using a 1 :500 molar enzyme to substrate ratio (reaction 17). X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

Figure 10 shows the deconvoluted mass spectrum of RL27_EVLFQGP_glucagon after overnight incubation at 4°C with RL9-HRV14 3C protease using a 1:20 molar enzyme to substrate ratio (reaction 18, control). X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

Figure 11 shows RL27_EVLFQGP_GLP-1(7-37, K34R) deconvoluted mass spectrum. X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

Figure 12 shows that RL27_EVLFQGP_GLP-1(7-37, K34R) deconvoluted mass spectrum. X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

Figure 13 shows RL27_EVLFQGP_GLP-1(7-37, K34R) deconvoluted mass spectrum. X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

Figure 14 shows RL27_EVLFQGP_GLP-1(7-37, K34R) deconvoluted mass spectrum. X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

Figure 15 shows that RL27_EVLFQGP_GLP-1(7-37, K34R) deconvoluted mass spectrum. X-axis: mass/charge ratio in Da. Y-axis: relative intensity.

describe

According to the first aspect of the present invention, a bifunctional fusion enzyme is provided, which comprises picornavirus 3C protease and the catalytic domain of XaaProDAP.

According to a second aspect of the present invention, there is provided a bifunctional fusion protease comprising a protein of the following formula:

X-Y-Z (I) or Z-Y-X (II)

in

X is a picornavirus 3C protease or a functional variant thereof;

Y is an optional linker;

Z is Xaa-Pro-dipeptidyl aminopeptidase (XaaProDAP) or a functional variant thereof;

Wherein said fusion protease has substantially no self-cleavage activity capable of impairing at least one of the two proteolytic activities.

The methods of the invention offer a number of advantages over previously described methods for the release of mature proteins from fusion proteins. For example, it has been unexpectedly found that very specific fusion proteolysis can be achieved, releasing the mature protein with the correct native N-terminal amino acid in high yield with no or minimal levels of associated impurities. The presence of any related impurities (ie proteins that resemble the mature protein in such a way that they have limited differences in chemical structure) is clearly undesirable as they are difficult and therefore expensive to remove during the manufacturing process. Additional embodiments have the advantage of enabling release of the mature protein from the fusion protein under reaction conditions with low temperature.

It has also been unexpectedly found that the bifunctional fusion protease of the present invention can be prepared by recombinant expression in E. coli. Often, it is difficult to express large proteins in E. coli without causing problems. However, the bifunctional fusion protease of the present invention can be produced by recombinant expression in E. coli, as shown in the disclosed examples of the present invention.

The inventors of the present invention aim to provide a fusion protease comprising functional XaaProDAP and functional picornavirus 3C protease. Such bifunctional fusion proteases should be expressible in microorganisms and should be stable during expression, purification and use to release the mature protein from the fusion protein. During the preparation of this bifunctional fusion protease, many technical challenges were encountered. First, HRV14 3C was found to be cleaved by itself by the HRV14 3C-XaaProDAP fusion protease, making the fusion protease unstable. Second, HRV14 3C also cleaves the HRV14 3C-XaaProDAP fusion protease internally in XaaProDAP from Lactococcus lactis at a site not recognized as a canonical HRV14 3C cleavage site. This also destabilizes the fusion protease. Again, when XaaProDAP is located at the C-terminus of the fusion protease, XaaProDAP from Lactococcus lactis can remove the dipeptide from the N-terminus of HRV14 3C – XaaProDAP fusion protease. Thus, the first fusion protease exhibited self-cleavage at three different sites, resulting in no activity and a challenge to be resolved if expression, purification, catalytic function, stability, or a combination of these of the bifunctional fusion protease were the cause Task.

When designing the bifunctional fusion protease of the present invention, the following steps can be carried out:

a) providing XaaProDAP or functional variants that do not have a QG subsequence available on the surface of the protein,

b) providing a picornavirus 3C protease or a functional variant thereof which, if located at the N-terminus of the bifunctional fusion protease, does not have a XaaProDAP cleavage site at its N-terminus and does not have a cleavage site enabling passage at its C-terminus cleavage to excise its own cleavage site, and

c) connecting XaaProDAP and picornavirus 3C protease via an optional amino acid linker sequence to form a bifunctional fusion protease that can be expressed by a single nucleic acid sequence.

It should be understood that the terms polypeptide, peptide and protein are used interchangeably in the present context. Likewise, amino acids are abbreviated to one-letter or three-letter names according to IUPAC nomenclature.

The bifunctional fusion protease of the present invention preferably exhibits sufficient activity at low temperatures (e.g. 2-10°C or 2-15°C), as this is desirable from an industrial manufacturing point of view, e.g. control microbial activity.

As used herein, "Xaa-Pro dipeptidyl aminopeptidase"("XaaProDAP") is intended to mean an enzyme having dipeptidase activity specific for an Xaa-Pro dipeptide, i.e., a scissile bond linking an Xaa-Pro dipeptide. C-terminus and N-terminus of target peptide or protein. According to the International Union of Biochemistry and Molecular Biology (IUBMB) enzyme nomenclature, XaaProDAP is classified as enzyme EC 3.4.14.11 from peptidase family S15 and enzyme EC 3.4.14.5 from peptidase family S9B. A non-limiting example of XaaProDAP is dipeptidyl-peptidase IV (DPP-IV) from mammals. Other non-limiting examples of XaaProDAP are Xaa-prolyl dipeptidyl aminopeptidases from cells such as those from Lactococcus lactis , Streptococcus thermophilus , Lactobacillus delbrueckii and porcine Streptococcus cus suis . The Xaa-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris CNCM 1-1631 has the following sequence:

MRFNHFSIVDKNFDEQLAELDQLGFRWSVFWDEKKILKDFLIQSPTDMTVLQANTELDVIEFLKSSIELDWEIFWNITLQLLDFVPNFDFEIGKATEFAKKLNLPQRDVEMTTETIISAFYYLLCSRRKSGMILVEHWVSEGLLPLDNHYHFFNDKSLATFDSSLLEREVVWVESPVDTEQKGKNDLIKIQIIRPKSTEKLPVVITASPYHLGINEKANDLALHEMNVDLEKKDSHKIHVQGKLPQKRPSETKELPIVDKAPYRFTHGWTYSLNDYFLTRGFASIYVAGVGTRGSNGFQTSGDYQQIYSMTAVIDWLNGRTRAYTSRKKTHEIKATWANGKVAMTGKSYLGTMAYGAATTGVDGLEVILAEAGISSWYNYYRENGLVRSPGGFPGEDLDVLAALTYSRNLDGADYLKGNDEYEKRLAEMTTALDRKSGDYNQFWHDRNYLINSDQVRADVLIVHGLQDWNVTPEQAYNFWQALPEGHAKHAFLHRGAHIYMNSWQSIDFSETINAYFSAKLLDRDLNLNLPPVILQENSKEQVWSAVSKFGGDDQLKLPLGKTAVSFAQFDNHYDDESFKKYSKDFNVFKKDLFENKANEAVIDLELPSELTINGPIELEIRLKLNDSKGLLSAQILDFGPKKRLEDKARVKDFKVLDRGRNFMLDDLVELPLVESPYQLVTKGFTNLQNKDLLTVSDLKADEWFTLKFELQPTIYHLEKADKLRVILYSTDFEHTVRDNRKVTYEIDLSQSKLIIPIESVKK (SEQ ID NO: 1).

XaaProDAP may be an enzyme that occurs naturally in eg bacteria or mammals, but it may also be a functional variant of such an enzyme. Non-limiting examples of functional variants are analogues, extended or truncated forms of naturally occurring XaaProDAP which retain the dipeptidase activity specific for the Xaa-Pro dipeptide.

The picornavirus 3C proteases (or protein 3C, Picornian 3C, or picornavirus 3C) are a group of cysteine proteases with a serine protease-like fold that are responsible for the production of mature proteases from precursor polyproteins in Picornaviridae viruses. viral protein.

As used herein, "picornavirus 3C protease" is intended to mean a protease from the Picornaviridae family, including functional variants thereof, that cleaves the peptide bond between the P1-P1´ Gln-Gly pair, where the scissile bond Linking Gln to Gly (where P1 and P1′ represent the first amino acid on the N-terminal and C-terminal sides of the scissile bond, respectively, according to common notation). Several picornavirus 3C proteases have an additional preference for Pro at P2', where P2' represents the second amino acid on the C-terminal side of the scissile bond. Enzymes with this substrate specificity are typically isolated from viruses of the enterovirus genus, which currently includes Coxsackie viruses. virus), Echovirus, enterovirus, poliovirus, and rhinovirus.这样的小RNA病毒3C蛋白酶的非限制性实例有：人鼻病毒14型3C (HRV14 3C)蛋白酶，其具有序列GPNTEFALSLLRKNIMTITTSKGEFTGLGIHDRVCVIPTHAQPGDDVLVNGQKIRVKDKYKLVDPENINLELTVLTLDRNEKFRDIRGFISEDLEGVDATLVVHSNNFTNTILEVGPVTMAGLINLSSTPTNRMIRYDYATKTGQCGGVLCATGKIFGIHVGGNGRQGFSAQLKKQYFVEKQ (SEQ ID NO: 2); Enterovirus 71 3C protease; Coxsackievirus A16 3C protease; Coxsackievirus B3 3C protease; Coxsackie mosaic Vigna mosaic virus type picornavirus 3C; and human poliovirus 3C protease. These 3C proteases are capable of releasing proteins with Gly-Pro at the N-terminus from large fusion proteins and can often be identified by having a naturally occurring Gly-Pro at their own native N-terminus. According to the invention, the picornavirus 3C protease may be a naturally occurring enzyme in the picornaviridae family, but it may also be a functional variant of such an enzyme. Non-limiting examples of functional variants are analogues, extended or truncated forms of the naturally occurring picornavirus 3C protease, which functional variants retain substrate specificity for the Gln-Gly pair.

As used herein, "substantially free of self-cleavage activity capable of impairing at least one of the two proteolytic activities" is intended to mean that, under expression conditions, purification conditions, storage conditions and manufacturing uses of precursors that cleave the target protein, The bifunctional fusion protease does not cleave itself, or cleaves itself only at a very slow rate, without hindering its intended use of cleaving the precursor of the target protein.

In one embodiment, "substantially free of self-cleavage activity capable of impairing at least one of the two proteolytic activities" is determined by the bifunctional fusion protease being sufficiently stable under the conditions of manufacture to cleave the precursor of the target protein .

In another embodiment, said fusion protease is determined to be substantially free of self-cleavage activity capable of impairing at least one of two proteolytic activities by determining that said bifunctional fusion protease is suitable for its intended use.

In another embodiment, determining said fusion protease that is substantially free of self-cleavage activity capable of impairing at least one of the two proteolytic activities is performed at a concentration of 0.5 mg/mL in 1x PBS buffer, pH It is determined that at least 50% of the bifunctional fusion protease is intact after incubating the bifunctional fusion protease at a temperature of 37° C. for 3 hours in 7.4.

In another embodiment, determining said fusion protease that is substantially free of self-cleavage activity capable of impairing at least one of the two proteolytic activities is performed at a concentration of 0.5 mg/mL in 1x PBS buffer, pH It is determined that at least 50% of the picornavirus 3C protease activity and XaaProDAP activity of the bifunctional fusion protease are intact after incubating the bifunctional fusion protease at a temperature of 37° C. for 3 hours in 7.4.

In another embodiment, determining said fusion protease that is substantially free of self-cleavage activity capable of impairing at least one of the two proteolytic activities is performed at a concentration of 0.5 mg/mL in 1x PBS buffer, pH It is determined that at least 80% of the picornavirus 3C protease activity and XaaProDAP activity of the bifunctional fusion protease are intact after incubating the bifunctional fusion protease at a temperature of 37° C. for 3 hours in 7.4.

In another embodiment, determining said fusion protease that is substantially free of self-cleavage activity capable of impairing at least one of the two proteolytic activities is performed at a concentration of 0.5 mg/mL in 1x PBS buffer, pH It is determined that at least 50% of the picornavirus 3C protease activity and XaaProDAP activity of the bifunctional fusion protease are intact after incubating the bifunctional fusion protease at a temperature of 4° C. for 24 hours in 7.4.

In another embodiment, determining said fusion protease that is substantially free of self-cleavage activity capable of impairing at least one of the two proteolytic activities is performed at a concentration of 0.5 mg/mL in 1x PBS buffer, pH It is determined that at least 80% of the picornavirus 3C protease activity and XaaProDAP activity of the bifunctional fusion protease are intact after incubating the bifunctional fusion protease at a temperature of 4° C. for 24 hours in 7.4. "Mature protein" as used herein is intended to mean a protein, peptide or polypeptide of interest, or an extended form thereof, which is cleavable at its N-terminus by XaaProDAP. Mature proteins typically exist as fusion proteins during their manufacture, eg, proteins comprising, in addition to the mature protein, a tag sequence, an optional linker sequence, and a picornavirus 3C protease site. Non-limiting examples of mature proteins are Glucagon, PYY(3-36), GLP-1(7-37), Arg34-GLP1(7-37), Arg34-GLP-1(9-37) and Arg34 - GLP-1(11-37). Commonly used single letter abbreviations for amino acid residues are used, eg, Arg34-GLP-1(7-37) is K34R-GLP-1(7-37) (also known as GLP-1(7-37, K34R)).

As used herein, "fusion protein" is intended to mean a hybrid protein expressible by a nucleic acid molecule comprising nucleotide sequences encoding at least two different proteins. For example, a fusion protein may comprise a tag protein fused to a protein having a pharmaceutical activity of interest. Fusion proteins are commonly used to enhance recombinant expression of therapeutic proteins and to enhance the recovery and purification of such proteins from cell culture, among other things. Fusion can also be used to combine the activities of two different enzymes into a single protein. Fusion proteins may also contain artificial sequences, such as linker sequences.

As used herein, "fusion protease" is intended to mean a hybrid protein expressible by a nucleic acid molecule comprising nucleotide sequences encoding at least two different proteins each having proteolytic activity. For example, a fusion protease can comprise two different proteases, such as an endopeptidase and an exoprotease. A fusion protease may also comprise, for example, a tag protein fused to two proteolytic proteins.

In one embodiment, the two different proteins comprised by the fusion protease exhibit two different proteolytic activities. In another embodiment, the two different proteins comprised by the fusion protease are proteases or functional variants thereof derived from different organisms.

The XaaProDAP protease has a protein structure comprising two alpha helices linked together by a large protein loop. This loop is exposed on the surface of the protein and is thus susceptible to cleavage by the picornavirus 3C protease, especially when the picornavirus 3C protease and XaaProDAP are contained in a bifunctional fusion protease. The loop of XaaProDAP connecting two small α-helices represents a highly conserved region among XaaProDAP proteases. In SEQ ID NO: 1, the loop is a subsequence spanning approximately 223 to 270 residues. The present inventors found that XaaProDAP is unstable when fused to HRV14 3C, which is caused by HRV14 3C cleavage of the QG subsequence at position 241-242. This was very unexpected since this loop does not contain a subsequence that is the cleavage site for the common picornavirus 3C protease. Therefore, this problem was solved by using functional variants of XaaProDAP that substituted the QG amino acid for other amino acids (eg ET).

As used herein, "fusion partner protein" or "fusion partner" is intended to mean a protein that is part of a fusion protein, ie one of at least two proteins comprised by the fusion protein. Non-limiting examples of fusion partner proteins are tag proteins and solubilizing domains, such as His6-tag, maltose binding protein, thioredoxin, and the like.

As used herein, "fusion enzyme" is intended to mean a fusion protein comprising at least two proteins that are both enzymes (meaning that the two proteins have covalently linked backbone sequences).

"Tag protein" or "tag" as used herein is intended to mean a protein which is linked to another protein to facilitate or enhance the manufacture of said other protein, for example to facilitate or enhance recombinant expression, recovery and/or or purification. Non-limiting examples of tagged proteins are His6-tag, glutathione S-transferase (GST), maltose binding protein (MBP), S. aureus protein A, biotinylated peptides and WO2006/108826 and WO2008/043847 Highly basic proteins from thermophilic bacteria as described in .

"Tag sequence" as used herein is intended to mean a sequence comprising a protein. The tag sequence may also optionally comprise additional sequences, such as linker sequences. Protein tags are peptide sequences genetically grafted onto recombinant proteins that can be removed by chemical reagents or by enzymatic methods (eg, proteolysis). Tags are attached to proteins for a variety of purposes, such as to facilitate protein expression or secretion from cells, to improve protein solubility, or to facilitate proper protein folding.

As used herein, "linker" is intended to mean an amino acid sequence commonly used to facilitate the function, folding or expression of a fusion protein. It is known to those skilled in the art that two proteins in the form of a fusion enzyme may interfere with each other's enzymatic activity, an interaction that can generally be eliminated or reduced by inserting a linker between the two enzyme sequences.

As used herein, "analogue" is intended to mean a protein derived from another protein by substitution, deletion and/or addition of one or more amino acid residues of that protein. Non-limiting examples of GLP-1(7-37) analogs are K34R-GLP-1(7-37) with residue 34 replaced by an arginine residue and residue 34 replaced by an arginine residue K34R-GLP-1(9-37) with amino acid residues 7-8 deleted (using the usual numbering of amino acid residues for GLP-1 peptides).

As used herein, "functional variant" is intended to mean a chemical variant of a protein that has an altered amino acid sequence, but retains substantially the same function as the original protein. Thus, a functional variant is generally a modified form of a protein in which as few modifications as necessary are introduced into the modified protein to obtain some desired property while retaining substantially the same function as the original protein. Non-limiting examples of functional variants are e.g. elongated proteins, truncated proteins, fusion proteins and the like. Non-limiting examples of functional variants of HRV14 3C are eg His6-tagged HRV14 3C, GST-tagged HRV14 3C and truncated (eg to not contain the N-terminal GP dipeptide) HRV14 3C. A non-limiting functional variant of GLP-1(7-37) is K34R-GLP-1(7-37).

In one embodiment, a functional variant of a protein comprises 1-2 amino acid substitutions, deletions or additions compared to said protein. In another embodiment, the functional variant comprises 1-5 amino acid substitutions, deletions or additions compared to said protein. In another embodiment, the functional variant comprises 1-15 amino acid substitutions, deletions or additions relative to the corresponding naturally occurring protein or naturally occurring subsequence of the protein.

"Solubilization domain" as used herein is intended to mean a protein which is part of a fusion protein and which serves to render the fusion protein more soluble under specific conditions than the protein of interest itself. Non-limiting examples of solubilizing domains are DsbC (thiol, disulfide bond interchanger), RL9 (ribosomal protein L9), MPB (maltose binding protein), NusA (transcription termination/ antitermin) and Trx (thioredoxin).

"Enzyme treatment" as used herein is intended to mean contacting a substrate protein with an enzyme that catalyzes at least one reaction involving said substrate protein. A common enzymatic treatment involves contacting the fusion protein with an enzyme with proteolytic activity to separate the two proteins that are constituents of the fusion protein.

According to a fourth aspect of the present invention, there is provided a bifunctional fusion protease of the present invention for the removal of N-terminal peptides or proteins from larger peptides or proteins in order to obtain mature proteins with the expected N-terminal aa residues. The larger peptide or protein is typically a fusion protein comprising the mature protein and one or more tag sequences to facilitate recombinant expression of the protein, proper folding, purification purposes, and the like.

In one embodiment, said larger peptide or protein is contacted with said bifunctional fusion protease under suitable reaction conditions for a time sufficient to release a majority of said N-terminal peptide. Reaction conditions can include, for example, a pH in the range of about 6.0 to about 9.0, about 7.0 to about 8.5, about 7.5 to about 8.5, about 8.0 to about 9.0, or about 6.0 to about 7.0. Reaction conditions may include temperatures ranging from about 0°C to about 50°C, from about 30°C to about 37°C, from about 0°C to about 15°C, from about 0°C to about 10°C, from about 2°C to about 10°C, From about 5°C to about 15°C, from about 0°C to about 5°C, or from about 2°C to about 8°C. In another embodiment, the reaction conditions include a pH ranging from about pH 7.5 to about pH 8.5 and a temperature ranging from about 4°C to about 10°C. In yet another embodiment, the reaction conditions include a reaction time ranging from about 1 minute to about 3 hours. In yet another embodiment, the reaction conditions include a reaction time ranging from about 3 hours to about 24 hours. In yet another embodiment, the reaction time ranges from about 3 hours to about 24 hours, from about 3 hours to about 16 hours, from about 6 hours to about 24 hours, from about 10 hours to about 16 hours. In another embodiment, the reaction conditions include an aqueous medium comprising phosphate buffered saline, eg, 50 mM sodium phosphate + 0.9% sodium chloride. Phosphate buffered saline (abbreviated PBS) is a commonly used buffered solution and is typically an aqueous based saline solution containing sodium phosphate, sodium chloride and (in some solutions) potassium chloride and potassium phosphate. A typical 1x PBS buffer used for the enzyme reaction in the present invention is (8.05 mM Na2HPO4x2H2O, 1,96 mM KH2PO4, 140 mM NaCl, pH 7.4).

Other buffers that can be used in the reaction medium may be TRIS (tris(hydroxymethyl)-aminomethane) or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffers.

In another embodiment, a bifunctional fusion protease is co-expressed with the larger peptide or protein for in vivo release of the protein of interest during expression in the host cell. In another embodiment, the larger peptide or protein is contacted with the bifunctional fusion protease after isolation of the two proteins from the host cell for their expression.

In another embodiment, said larger peptide or protein is selected from comprising peptides or proteins comprising a peptide selected from the group consisting of: GLP-1 (glucagon-like peptide 1), glucagon, peptide YY (PYY) and amyloid peptide (amylin) and their functional variants.

In yet another embodiment, the larger peptide or protein is less than 200 amino acid residues, less than 150 amino acid residues, less than 100 amino acid residues, or less than 60 amino acid residues in size.

"Application" means a sample comprising a fusion protein that is applied to a purification column.

"Flow through" means the portion of the application that contains host cell proteins and contaminants that do not bind to the purification column.

"Major peak" refers to the peak in the purified chromatogram that has the highest UV intensity and contains the fusion protein.

"UV280 intensity" is the absorbance measured in milliabsorbance units at a wavelength of 280 nm at which a protein absorbs.

"UV215" is the absorbance measured in milliabsorbance units at a wavelength of 215 nm at which a protein absorbs.

"IPTG" is isopropyl-ß-D-thiogalactopyranoside.

TIC is Total Ion Count.

HPLC is High Performance Liquid Chromatography.

LC-MS refers to Liquid Chromatography Mass Spectrometry.

"% Purity" is defined as amount of specific protein/(amount of specific protein + amount of contaminant) X 100.

SDS-PAGE is sodium dodecyl sulfate polyacrylamide gel current.

According to a third aspect of the present invention, there is provided a method for preparing the bifunctional fusion protease of the present invention, which comprises recombinantly expressing a protein comprising the bifunctional fusion protease in a host cell and subsequently isolating the bifunctional fusion protease.

In one embodiment, the method for preparing a bifunctional fusion protease includes Escherichia coli as the host cell.

In another embodiment, the method for preparing a bifunctional fusion protease comprises isolating the bifunctional fusion protease into a soluble protein.

In another embodiment, the method for making a bifunctional fusion protease comprises isolating said bifunctional fusion protease into a soluble protein without using a refolding step.

In another embodiment, the method for preparing a bifunctional fusion protease comprises a bifunctional fusion protease having the formula (I) shown in Embodiment 2, that is, the picornavirus 3C protease or a functional variant thereof is located in the N-terminal portion of a bifunctional fusion protease.

Bifunctional fusion proteases can be produced by recombinant protein technology. Typically, the cloned wild-type picornavirus 3C protease and the cloned wild-type XaaProDAP nucleic acid sequence or functional variant thereof are modified to encode the desired fusion protein. Such modifications include in-frame fusion of nucleic acid sequences encoding two or more proteins to be expressed as fusion proteins. Such fusion proteins may be bifunctional fusion proteases, with or without a linker peptide, and bifunctional fusion proteases fused to a tag, such as a His-tag or a solubilizing domain (eg DsbC, RL9, MBP, NusA or Trx). The modified sequence is then inserted into an expression vector, and then transformed or transfected into an expression host cell.

The nucleic acid construct encoding the bifunctional fusion protease may suitably be of genomic, cDNA or synthetic origin. Amino acid sequence changes are accomplished by altering the genetic code using known techniques.

Usually, the DNA sequence encoding the bifunctional fusion protease is inserted into any recombinant vector, which can conveniently carry out recombinant DNA procedures, and the choice of the vector often depends on the host cell into which it is to be introduced. Thus, a vector may be an autonomously replicating vector, ie a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, eg a plasmid. Alternatively, the vector may be a vector which, when introduced into a host cell, is integrated into the genome of the host cell and replicated together with the chromosome into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequence encoding the bifunctional fusion protease is operably linked to additional segments required for DNA transcription. The term "operably linked" denotes an arrangement of segments such that they function in harmony for their intended purpose, eg, transcription begins at a promoter and progresses through a DNA sequence encoding a polypeptide until it terminates at a terminator.

Accordingly, expression vectors for expression of bifunctional fusion proteases will contain a promoter capable of initiating and directing transcription of the cloned gene or cDNA. The promoter may be any DNA sequence that exhibits transcriptional activity in the host cell of choice and may be derived from a gene encoding a protein either homologous or heterologous to the host cell.

In addition, expression vectors for bifunctional fusion proteases will also contain terminator sequences, which are sequences recognized by the host cell to terminate transcription. A terminator sequence is operably linked to the 3' end of a nucleic acid sequence encoding a polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.

The expression of the bifunctional fusion protease can be targeted for intracellular expression in the cytosol of the host cell, or directed to the secretory pathway to reach the culture medium extracellularly.

Intracellular expression is the default route, requiring the expression vector to have a DNA sequence comprising a promoter, followed by a DNA sequence encoding a bifunctional fusion protease polypeptide, and then a terminator.

In order to direct the bifunctional fusion protease to the secretory pathway of the host cell, a secretory signal sequence (also known as signal peptide or presequence) is required as an N-terminal extension of the bifunctional fusion protease. The DNA sequence encoding the signal peptide is connected to the 5' end of the DNA sequence encoding the bifunctional fusion protease in the correct reading frame. The signal peptide may be one normally associated with the protein, or may be from a gene encoding another secreted protein.

Procedures for ligating DNA sequences encoding bifunctional fusion proteases, promoters, terminators and/or secretion signal sequences, respectively, and for inserting them into suitable vectors containing the information necessary for replication are within the skill of the artisan Well known (see for example Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

The host cell into which the DNA sequence encoding the bifunctional fusion protease is introduced can be any cell capable of expressing the bifunctional fusion protease intracellularly or extracellularly. If post-translational modifications are desired, suitable host cells include yeast, fungi, insects, and higher eukaryotic cells, such as mammalian cells.

bacterial expression

For expression in E. coli , examples of suitable promoters for directing transcription of nucleic acid constructs in bacterial host cells are those available from the lac operon, trp operon and their hybrids trc and tac , all from Escherichia coli ( DeBoer et al ., 1983, Proceedings of the National Academy of Sciences USA 80:21-25). Other even stronger promoters for use in E. coli are the phage promoters from T7 and T5 phage. The T7 promoter requires the presence of T7 polymerase in the E. coli host (Studier and Moffatt, J. Mol. Biol. 189 , 113, (1986)). All of these promoters are regulated by IPTG, lactose or tryptophan induction to initiate transcription at strategic time points during bacterial growth. E. coli also has strong promoters for continuous expression, such as the synthetic promoter for expression of hGH in Dalbøge et al., 1987, Biotechnology 5 , 161-164.

For expression in Bacillus , suitable examples are promoters from the following: Bacillus subtilis fructan sucrase gene ( sacB ), Bacillus licheniformis α-amylase gene ( amyL ), Bacillus stearothermophilus ( Bacillus stearothermophilus ) maltogenic amylase gene ( amyM ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) α-amylase gene ( amyQ ), Bacillus licheniformis penicillinase gene ( penP ), Bacillus subtilis xylA and xylB genes. Further promoters are described in: "Useful proteins from recombinant bacteria", Scientific American , 1980, 242:74-94; and Sambrook et al., 1989, supra.

For E. coli, effective signal peptide coding regions for bacterial host cells have signal peptides obtained from the following genes: DegP , OmpA , OmpF , OmpT , PhoA , and endotoxin STII, all from E. coli. For Bacillus, the signal peptide region was obtained from Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutropease (nprT, nprS, nprM) and Bacillus subtilis prsA. Further signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57: 109-137. For both E. coli and Bacillus, signal peptides can be generated de novo according to the rules outlined in the algorithm SignalP (Nielsen et al., 1997, Protein Eng. 10 , 1-6., Emanuelsen et al., 2007, Nature Protocols 2 , 953-971) . Adapt the signal sequence to the given environment and check the SignalP score.

Examples of strong transcriptional terminators are the aspartase aspA in the Thiofusion expression system, the T7 gene 10 terminator in PET vectors (Studier et al.) and the terminators of the ribosomal RNA genes rrnA , rrnD .

Examples of preferred expression hosts are Escherichia coli K12 W3110, Escherichia coli K12 with B, MC1061 marker, and Escherichia coli B BL21 DE3, which contains T7 polymerase by lysogenization with bacteriophage. These hosts can be selected with antibiotics when transformed with plasmids for expression. For antibiotic-free selection, preferred hosts are e.g. E. coli B BL21 DE3 3xKO with deletions of the 2 D,L-alanine racemase genes Δalr , ΔdadX and deletions unique to E. coli B and often associated with the pathogen Property-related group II capsular gene cluster Δ (kpsM-kpsF) . Deletion of the group II gene cluster made the safety category of E. coli B BL21 DE3 3xKO the same as that of E. coli K12. The selection was based on not requiring D-alanine, provided by the alr gene inserted in the expression plasmid in place of the AmpR gene.

Once the bifunctional fusion protease is expressed in the host organism, it can be recovered and purified to the desired purity by conventional techniques. Non-limiting examples of such conventional recovery and purification techniques are centrifugation, dissolution, filtration, precipitation, ion exchange chromatography, immobilized metal affinity chromatography (IMAC), RP-HPLC, gel filtration and freeze drying.

Examples of recombinant expression and purification of HRV14 3C can be found eg in: Cordingley et al., J. Virol. 1989, 63, pp5037-5045; Birch et al., Protein Expr Purif., 1995, 6, pp609-618; and WO2008/043847.

Examples of microbial expression and purification of XaaProDAP by Lactococcus lactis can be found eg in: Chich et al., Anal. Biochem, 1995, 224, pp 245-249; and Xin et al., Protein Expr. Purif. 2002, 24, pp530-538.

The invention is further illustrated by the following non-limiting embodiments:

1. A bifunctional fusion enzyme comprising the catalytic domain of picornavirus 3C protease and XaaProDAP.

2. The bifunctional fusion protease of embodiment 1, it comprises the proteoprotein of following formula:

X-Y-Z (I) or Z-Y-X (II)

in

X is a picornavirus 3C protease or a functional variant thereof;

Y is an optional linker;

Z is Xaa-Pro-dipeptidyl aminopeptidase (XaaProDAP) or a functional variant thereof;

Wherein said fusion protease has substantially no self-cleavage activity capable of impairing at least one of the two proteolytic activities.

3. The bifunctional fusion protease according to any one of embodiments 1-2, which has formula (I), that is, the picornavirus 3C protease or its functional variant is located at the N-terminal part of the bifunctional fusion protease.

4. The bifunctional fusion protease of any one of embodiments 1-3, wherein X is a rhinovirus protease or a functional variant thereof.

5. The bifunctional fusion protease of any one of embodiments 1-3, wherein X is a picornavirus protease or a functional variant thereof.

6. The bifunctional fusion protease according to any one of embodiments 1-4, wherein X is HRV14 3C or a functional variant thereof.

7. The bifunctional fusion protease according to any one of embodiments 1-6, wherein X comprises SEQ ID NO: 2 or a functional variant thereof.

8. The bifunctional fusion protease according to any _one of embodiments 5-6, wherein X is X selected from P2X1 _- SEQ ID NO:2 or G1P-SEQ ID NO: of genetically encoded amino acid residues other than P 2 or a functional variant thereof.

9. The bifunctional fusion protease according to any one of embodiments 5-6, wherein X is CVB3 3C or a functional variant thereof.

10. The bifunctional fusion protease of embodiment 5, wherein X comprises SEQ ID NO: 23 or a functional variant thereof.

11. The bifunctional fusion protease according to any one of embodiments 1-10, wherein X is a C-terminal truncated functional picornavirus 3C protease or a functional variant thereof.

12. The bifunctional fusion protease of embodiment 11, wherein the C-terminal truncated functional picornavirus 3C protease is truncated by no more than 20 amino acid residues, such as by no more than 10 amino acid residues, such as No more than 5 amino acid residues, such as no more than 2 amino acid residues.

13. The bifunctional fusion protease of any one of embodiments 1-12, wherein X is an enzyme from a virus selected from the group consisting of enterovirus, coxsackie virus, echovirus, cowpea mosaic cowpea mosaic virus, Rhinoviruses and polioviruses, or functional variants thereof.

14. The bifunctional fusion protease according to any one of embodiments 1-13, wherein Z is the E.C. 3.4.14.11 enzyme or a functional variant thereof.

15. The bifunctional fusion protease of embodiment 14, wherein Z is an enzyme from lactic acid bacteria or a functional variant thereof.

16. The bifunctional fusion protease of embodiment 15, wherein Z is derived from Lactococcus spp . , Streptococcus spp. , Lactobacillus spp. , Bifidobacterium spp. Enzymes or functional variants thereof.

17. The bifunctional fusion protease according to any one of embodiments 1-16, wherein Z is SEQ ID NO: 1 or a functional variant thereof.

18. The bifunctional fusion protease according to any one of embodiments 1-14, wherein Z is an enzyme from Bacillus spp . or a functional variant thereof.

19. The bifunctional fusion protease of any one of embodiments 1-16, wherein Z is an enzyme from Strept ococ cus suis or a functional variant thereof.

20. The bifunctional fusion protease of embodiment 17, wherein Z is SEQ ID NO: 24 or a functional variant thereof.

21. The bifunctional fusion protease according to any one of embodiments 1-17, wherein Z is an enzyme from Lactococcus lactis or a functional variant thereof.

22. The bifunctional fusion protease of any one of embodiments 1-13, wherein said Z is an E.C. 3.4.14.5 enzyme or a functional variant thereof.

23. The bifunctional fusion protease according to any one of embodiments 1-22, wherein Z is a protein with an exposed loop connecting two α-helices.

24. The bifunctional fusion protease of embodiment 23, wherein said loop does not comprise any QG subsequences.

25. The bifunctional fusion protease according to any one of embodiments 23-24, wherein said loop does not comprise any of the following subsequences: QS, QI, QN, QA, and QT.

26. The bifunctional fusion protease according to any one of embodiments 23-25, wherein said loop is a sequence spanning amino acid residues 223-270 in SEQ ID NO: 1 .

27. The bifunctional fusion protease according to any one of embodiments 23-26, wherein said loop is a sequence in XaaProDAP corresponding to the sequence spanning amino acid residues 223-270 in SEQ ID NO: 1 .

28. The bifunctional fusion protease according to any one of embodiments 23-27, wherein said loop is a sequence having at least 70% amino acid identity to a sequence spanning residues 223-270 of amino acid residues in SEQ ID NO: 1 .

29. The bifunctional fusion protease of any one of embodiments 1-28, wherein Z comprises no more than 1 QG subsequence.

30. The bifunctional fusion protease of any one of embodiments 1-29, wherein Z does not comprise any QG subsequences.

31. The bifunctional fusion protease according to any one of embodiments 1-17, wherein Z comprises at least 1 substitution, addition or deletion of amino acid residues in Q241-G242.

32. The bifunctional fusion protease of embodiment 31, wherein Z comprises the substitutions Q241E, G242T.

33. The bifunctional fusion protease according to any one of embodiments 1-32, wherein the second amino acid residue from the N-terminus in the fusion protease is not P.

34. The bifunctional fusion protease according to any one of embodiments 1-33, wherein the second amino acid residue from the N-terminus in the fusion protease is not G, A and T.

35. The bifunctional fusion protease according to any one of embodiments 1-33, wherein the N-terminus of the fusion protease has the amino acid sequence MX ₁ P, wherein X ₁ is an aminopeptidase such that the MX ₁ P sequence is methionine Amino acids that are poor substrates.

36. The bifunctional fusion protease according to any one of embodiments 1-34, wherein the N-terminal amino acid residue of said bifunctional fusion protease is P.

37. The bifunctional fusion protease of embodiment 36, wherein the second amino acid residue from the N-terminus in the fusion protease is not P, G, A or T.

38. The bifunctional fusion protease of any one of embodiments 1-37, which does not comprise a linker Y.

39. The bifunctional fusion protease of any one of embodiments 1-37, comprising a linker Y.

40. The bifunctional fusion protease of embodiment 39, wherein said linker Y is 2-100 amino acid residues in length.

41. The bifunctional fusion protease according to any one of embodiments 39-40, wherein said linker Y is 2-50 amino acid residues in length.

42. The bifunctional fusion protease according to any one of embodiments 39-41, wherein said linker Y is 2-25 amino acid residues in length.

43. The bifunctional fusion protease according to any one of embodiments 39-42, wherein said linker Y is 2-15 amino acid residues in length.

44. The bifunctional fusion protease of any one of embodiments 39-41, wherein Y is about 5 to about 50 amino acid residues in length.

45. The bifunctional fusion protease of any one of embodiments 38-39, wherein Y is about 5 to about 15 amino acid residues in length.

46. The bifunctional fusion protease according to any one of embodiments 39-45, wherein Y does not comprise a Cys residue.

47. The bifunctional fusion protease according to any one of embodiments 39-46, wherein Y does not comprise a Gln residue.

48. The bifunctional fusion protease according to any one of embodiments 39-47, wherein Y comprises only the following amino acid residues: G, S, A, L, P and T.

49. The bifunctional fusion protease according to any one of embodiments 39-48, wherein Y is selected from SEQ ID NO: 3, 4 and 12.

50. The bifunctional fusion protease according to any one of embodiments 1-49, which is of formula (I), ie the picornavirus 3C protease or a functional variant thereof is located at the N-terminal portion of the bifunctional fusion protease.

51. The bifunctional fusion protease of embodiment 50, wherein X does not have the C-terminal amino acid residue Q.

52. The bifunctional fusion protease according to any one of embodiments 1-49, which is of formula (II), ie the picornavirus 3C protease or a functional variant thereof is located at the C-terminal portion of the bifunctional fusion protease.

53. The bifunctional fusion protease of any one of embodiments 1-52, comprising a tag protein linked to the N-terminus.

54. The bifunctional fusion protease according to embodiment 53, wherein said tag protein is selected from the group consisting of a His-tag, a solubilization domain, and a His-tag solubilization domain.

55. The bifunctional fusion protease according to any one of embodiments 1-54, wherein said functional variant comprises: 1-2 amino acid substitutions, deletions or additions relative to the corresponding naturally occurring protein or naturally occurring subsequence, Or 1-5 amino acid substitutions, deletions or additions, or 1-15 amino acid substitutions, deletions or additions.

56. The bifunctional fusion protease according to any one of embodiments 1-55, wherein said fusion protease is determined to be substantially free of self-cleavage activity capable of impairing at least one of the two proteolytic activities by said bifunctional fusion protease Functional fusion proteases suitable for their intended use are determined.

57. The bifunctional fusion protease according to any one of embodiments 1-55, wherein said fusion protease is determined to be substantially free of self-cleavage activity capable of impairing at least one of the two proteolytic activities by measuring The mg/mL concentration was determined when at least 50% of the bifunctional fusion protease was intact after incubating the bifunctional fusion protease in 1x PBS buffer, pH 7.4 at a temperature of 37°C for 3 hours.

58. The bifunctional fusion protease according to any one of embodiments 1-55, wherein said fusion protease is determined to be substantially free of self-cleavage activity capable of impairing at least one of the two proteolytic activities by measuring The mg/mL concentration was determined by incubating the bifunctional fusion protease in 1x PBS buffer, pH 7.4 at a temperature of 37°C for 3 hours after at least 50% of the picornavirus 3C protease activity and XaaProDAP activity of the bifunctional fusion protease were intact .

59. The bifunctional fusion protease of embodiment 58, wherein at least 80% of the bifunctional fusion protease is present after incubating said bifunctional fusion protease at a temperature of 37°C for 3 hours at a concentration of 0.5 mg/mL in 1x PBS buffer, pH 7.4 The picornavirus 3C protease activity and XaaProDAP activity are intact.

60. The bifunctional fusion protease according to any one of embodiments 1-55, wherein said fusion protease is determined to be substantially free of self-cleavage activity capable of impairing at least one of the two proteolytic activities by performing the fusion at 0.5 The mg/mL concentration was determined by incubating the bifunctional fusion protease in 1x PBS buffer, pH 7.4 at a temperature of 4°C for 24 hours after at least 50% of the picornavirus 3C protease activity and XaaProDAP activity of the bifunctional fusion protease were intact .

61. The bifunctional fusion protease of embodiment 60, wherein at least 80% of the bifunctional fusion protease is present after incubating said bifunctional fusion protease at a temperature of 4° C. for 24 hours at a concentration of 0.5 mg/mL in 1× PBS buffer, pH 7.4 The picornavirus 3C protease activity and XaaProDAP activity are intact.

62. A method for preparing the bifunctional fusion protease of any one of embodiments 1-61, comprising recombinantly expressing a protein comprising the bifunctional fusion protease in a host cell and subsequently isolating the bifunctional fusion protease.

63. The method of embodiment 62, wherein the host cell is E. coli.

64. The method of any one of embodiments 62-63, wherein the bifunctional fusion protease is isolated as a soluble protein.

65. The method of any one of embodiments 62-64, wherein the bifunctional fusion protease is isolated as a soluble protein using a refolding step.

66. The method according to any one of embodiments 62-65, wherein the bifunctional fusion protease has the formula (I) shown in embodiment 2, that is, the picornavirus 3C protease or a functional variant thereof is located in the bifunctional fusion protease N-terminal part of functional fusion protease.

67. Use of the bifunctional fusion protease of any one of embodiments 1-66 for the removal of N-terminal peptides or proteins from larger peptides or proteins.

68. The use of embodiment 67, wherein said larger peptide or protein is contacted with said bifunctional fusion protease under suitable reaction conditions for a time sufficient to release a majority of said N-terminal peptide.

69. The use according to any one of embodiments 67-68, wherein the bifunctional fusion protease is co-expressed with said larger peptide or protein for in vivo release of the protein of interest during expression in the host cell.

70. The use according to any one of embodiments 67-68, wherein said larger peptide or protein is contacted with said bifunctional fusion protease after isolation of the two proteins from the host cell used for their expression.

71. The use of any one of embodiments 67-70, wherein the larger peptide or protein is selected from a peptide or protein comprising a peptide selected from: GLP-1, glucagon, PYY, amyloid Peptides and their functional variants.

72. The use of any one of embodiments 67-71, wherein the larger peptide or protein is less than 200 amino acid residues, less than 150 amino acid residues, less than 100 residues, or less than 60 amino acid residues.

Example

Example 1

HRV14/XaaProDAP or XaaProDAP/HRV14 Plasmid construction and expression of variants

The pET system was used to express enzymes because this system provides a robust method of expressing proteins in E. coli. In pET vectors, target genes are cloned under the control of strong phage T7 transcription and release signals, and expression is induced by providing a source of T7 RNA polymerase in the host cell.

The E. coli expression plasmid (pET22b, Novagen) encodes a bifunctional fusion protease comprising a fusion of HRV14 3C and L. lactis XaaProDAP sequences. In one set of constructs, the HRV14 3C portion was located at the N-terminus of the XaaProDAP sequence using an intervening linker GGSGGSGGS (SEQ ID NO: 3 ) separates the two domains (Table 1).

Table 1. pET22b plasmid construct encoding NH2-HRV14 3C-XaaProDAP-COOH fusion protease

In another set of plasmids encoding fusion proteases, the HRV14 3C portion was placed C-terminal to the XaaProDAP sequence with an intervening linker GSSGSGGSG (SEQ ID NO: 4) separating the two domains.

Table 2. pET22b plasmid construct encoding NH2-XaaProDAP-HRV14 3C-COOH fusion protease

Fusion partners that enhance expression, purification or solubility are placed at the N-terminus of the two bifunctional protease variants. The fusion partner is designed to contain a His6 tag (located at the N- or C-terminus of the fusion partner sequence) encoding a flexible Gly-Ser-rich linker sequence comprising the Hepatitis A virus introduced near the N-terminus of the bifunctional protease sequence 3C protease (HAV) cleavage site with the sequence GGSSGSGSELRTQS (SEQ ID NO: 22) to allow enzymatic separation of the fusion partner from the protease moiety, if desired.

The gene fragments encoding the fusion proteases described in Tables 1 and 2 were codon-optimized for expression in E. coli and prepared by gene synthesis (GenScript). The plasmid constructs detailed in Tables 1 and 2 were generated by inserting the synthetic gene fragment into the pET22b vector using standard cloning techniques (obtained from GenScript) known to those skilled in the art.

Evaluation of fusion protease variants by small-scale expression and purification

The expression plasmid was transformed into Escherichia coli BL21(DE3) (Novagen) and expressed on a small scale.

E. coli BL21(DE3) was transformed with the plasmid using a Heat Shock based procedure at 42°C according to the manufacturer. Transformed cells were plated onto LB agar plates and incubated overnight at 37°C with 10 mg/L ampicillin. Overnight Terrific broth (TB) cultures containing 0.5% glucose and 50 mg/L carbenicillin containing each transformant were prepared at 30°C and shaken at 700 rpm using a Glas-Col shaker (Glas-Col). 20 µL of an overnight culture of each transformant was used to inoculate 0.95 µL of TB medium containing 50 mg/L carbenicillin in 96 deep well plates (2 ml), and the transformants were propagated overnight at 700 rpm. Expression cultures were incubated at 37°C until OD600 reached 1.5. The culture was then cooled to 20°C and protein induction was performed overnight with 0.3 mM IPTG. The pellet containing expressed protein was harvested by centrifugation at 1800xG.

Purification Screening: Small-scale purification using IMAC resins to evaluate combined expression and purification potential and integrity of proteases. Briefly, 250 µL of lysis buffer (50 mM NaPO4, 300 mM NaCl, 10 mM imidazole, 10mg/ml lysozyme, 250U/µL Benzoase and 10% DDM (dodecyl maltoside)) was added to each pellet , using freeze/thaw cycles to lyse cells. Debris was removed by centrifugation and the supernatant was filtered (0.45 µm) and transferred to a 1.2 µm filter plate containing Ni2+-supplemented Sepharose Fast Flow (prepared by washing 30 µL of a 50% slurry in 20% EtOH) (GE Healthcare). The supernatant was incubated for 20 minutes to bind protein by shaking with the resin at 400 rpm, and solutes were removed by gentle centrifugation at 100 xg for 1 minute. The resin was washed with 50 mM sodium phosphate, 300 mM NaCl, 30 mM imidazole, pH 7.5 by gentle mixing and dried by centrifugation. To elute the protein, 40 µL of wash buffer (50 mM sodium phosphate, 300 mM NaCl, 300 mM imidazole) was added to the resin, shaken at 400 rpm for 10 min, and the eluate containing partially purified enzyme was collected.

Whole lysates from pellets expressed by fusion protease variants were analyzed by SDS-PAGE. For all fusion protease variants described in Tables 1 or 2, no significant amount of full-length protein was observed. However, distinctly sized bands were observed for several fusion protease variants. SDS-PAGE analysis of IMAC-purified samples was consistent with these observations, as it also did not show production of the full-length protease, rather, a band of smaller size. Observations suggest that the fusion protease is truncated or degraded during expression and/or after capture onto IMAC resin. Since distinct bands were observed for several fusion protease variants, and expression levels appeared to be significantly based on gel band intensities, the absence of the full-length protein was more precisely due to undesired specific positions in the fusion protease hydrolysis, resulting in significant truncation of the fusion protease.

fusion protease LC-MS analyze

Possible cleavage sites that could account for the observed presence of truncated forms of the fusion protease were detected by mass spectrometry using a MaXis Impact ultra-high resolution time-of-flight (UHR-TOF) mass spectrometer (Bruker Daltonics) equipped with a Dionex UltiMate3000™ liquid A phase chromatograph (Dionex), according to the manufacturer's instructions, allowed diode array measurements at UV215 nm with typical settings. Enzymes were separated on a Waters Aquity BEH300 C4 reversed-phase 1.0 X 100 mm column with a pore size of 1.7 µm, using a column temperature of 45°C and a flow rate of 0.2 ml/min. The solvents used are as follows:

Solvent A: 0.1% formic acid H2O solution

Solvent B: 99.9% MeCN, 0.1% formic acid (v/v)

Liquid chromatography was performed using the following gradient to separate the enzymatic digests.

Time (minutes) %A %B

0 90 10

2 90 10

10 10 90

11 10 90

12 90 10

13 90 10

14 50 50

The recorded mass spectra were deconvoluted and analyzed with Bruker Compass data analysis software version 4.1 (Bruker Daltonics), according to the manufacturer's instructions, covering the mass range and resolution (>10.000) of 10.000 Da-140.000 Da. The UV215 nm chromatogram and the total ion count (TIC) chromatogram were evaluated in parallel to ensure agreement between the acquired MS data and the UV215 nm trace of the peptide. Experimentally determined masses shown refer to average isotopic masses and mass spectral data were obtained with mass accuracy better than 200 ppm.

When protease 12756 was analyzed, a mass of 22241.54 Da was detected. This mass corresponds to the mass of the His6 fusion partner (SEQ ID NO: 5) and HRV14 3C domain (SEQ ID NO: 2) (calculated mass 22242.27 Da). Thus, there is a cleavage site between the Gln/Gly linking the C-terminus of the HRV14 3C domain (SEQ ID NO: 2) and the N-terminus of the linker (SEQ ID NO: 3). This suggests that the 3C protease is able to excise itself from the fusion protease of HRV14 3C fused to the N-terminus of XaaProDAP in a manner similar to that reported for its native viral polyprotein. This was This was also observed for fusion proteases 12757, 12758, 12760 and 12761, where the size variation corresponds to the difference in the size of the N-terminal fusion partners used.

Example 2

remove NH2-HRV14-XaaProDAP-COOH fusion protease HRV14 in the domain C- the end Q182

For the fusion protease variants shown in Table 1, the size of the observed fragment often corresponds to the fusion partner plus HRV14 3C domain sequence. To remove this possibility of cleavage, a new linker was designed to replace the initial GS linker (SEQ ID NO:3) between the HRV14 3C and XaaProDAP domains in fusion proteases containing His6, RL9 or Trx fusion partners (Table 1, embodiment 1). The Gln/Gly cleavage site linking the HRV14 enzyme and the start of SEQ ID NO: 3 was replaced with Ser-Gly.因此，除去了HRV14 3C蛋白酶结构域中的最后一个氨基酸(Gln182)，得到具有下述序列的des182-HRV14 3C：GPNTEFALSLLRKNIMTITTSKGEFTGLGIHDRVCVIPTHAQPGDDVLVNGQKIRVKDKYKLVDPENINLELTVLTLDRNEKFRDIRGFISEDLEGVDATLVVHSNNFTNTILEVGPVTMAGLINLSSTPTNRMIRYDYATKTGQCGGVLCATGKIFGIHVGGNGRQGFSAQLKKQYFVEK (SEQ ID NO: 11), and removed the initial Gly of the linker (SEQ ID NO: 3), because this site represents the cleavage site of 3C protease. However, the linker between the HRV14 domain (SEQ ID NO: 11) and the XaaProDAP domain was replaced by SGSGGSGGSGS (SEQ ID NO: 12). The new fusion protease variants are shown in Table 3:

Table 3. pET22b plasmid construct encoding des182HRV14 3C-XaaProDAP fusion protease

Small scale expression and purification of these constructs was performed as described in Example 1. SDS-PAGE of samples from IMAC purification showed that at this point two clearly visible and predominant bands at approximately 50-60 kDa appeared for the three fusion protease variants, indicating that the full-length protease was cleaved into two fragments. LC-MS analysis was performed as described in Example 1 to map the cleavage site. Analysis of Protease 20177 showed that the fusion protease variant was cut into two large bands, each with a mass of 51091.27 Da and 59773.49 Da. These masses confirm that another cleavage site occurs between Gln241 and Gly242 of the XaaProDAP sequence (SEQ ID NO: 1), as the determined masses agree with the calculated masses of these fragments, as deduced from the protease 20177 amino acid sequence (respectively are 51092.15 Da and 59772.43 Da). By analyzing the deconvoluted spectra of all three constructs shown in Table 3, the exact same cleavage site was clearly observed, thus indicating that this site is highly sensitive, whereas the N-terminal fusion partner used irrelevant. In evaluating the available 3D structures (Rigolet et al., Structure, 10, pp 1384-1394), it was determined that the cleavage site exists in the very large α-helix linking two small α-helices in the catalytic domain of Lactococcus lactis XaaProDAP middle of the loop (spanning approximately amino acid residues 223-270). This loop is highly exposed and thus sensitive to cleavage, and the Q/G sequence suggests that the 3C protease itself is responsible for this cleavage. Another, less predominant, non-desired cleavage site was observed at the Gln/Ser position in the C-terminal HAV protease (ELRTQ/S) cleavage site of the fusion partner, which was also identified by analysis of IMAC-purified samples. detection.

Example 3A

Design Full Length Dual Function NH2-HRV14-XaaProDAP-COOH protease

To remove the cleavage site observed between Gln241 and Gly242 of the XaaProDAP sequence in Example 2, these two amino acids were substituted with Glu241 and Thr242. The Glu241-Thr242 substitution was chosen as an alternative to Gln241-Gly242 because of its presence as a natural amino acid change based on homology searches of XaaProDAP orthologs from different isolates of Lactococcus lactis. Since the undesired cleavage was also present at the HAV site at the C-terminus of the fusion partner, the HAV site was replaced with a small GS-containing sequence. These fusion partners have the sequence MHHHHHHGGSSGSGSGSGSGS (SEQ ID NO: 13), MKVILLRDVPKIGKKGEIKEVSDGYARNYLIPRGFAKEYTEGLERAIKHEKEIEKRKKEREREESEKILKELKKRTHVVKVKAGEGGKIFGAVTAATVAEEISKTTGLKLDKRWFKLDKPIKELGEYSLEVGSLPGGVKGSSHS (SEQ ID NO: 14) and MHHHHHHGSGSGSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGGSSGSGSGSGSGS (SEQ ID NO: 15).

A plasmid construct (Genscript) was obtained containing the Q241E, G242T substitutions with the HAV site removed by the linker in front of the HRV14 3C domain. The designs and tests are shown in Table 4.

Small scale expression and IMAC purification of the novel fusion protease constructs was performed as described in Example 1. As observed by SDS-PAGE analysis, the Q241E and G242T substitutions apparently prevented the fusion protease from being cleaved into two parts. For all three constructs, very strong gel bands around 100-120 kDa were observed, indicating that the Q241E, G242T substitutions resulted in a soluble and intact full-length fusion protease comprising the HRV14 3C domain and the XaaProDAP structure domain both. In this experiment, the benefit of removing the ELRTQ site (by substituting with GSGSG) was less pronounced.

LC-MS was performed on the fusion protease variants in Table 4 as described in Example 1 and confirmed the observations from SDS-PAGE. The determined masses of proteases 20986, 20988 and 20990 were 110604.97 Da, 127867.76 Da and 122607.21 Da, respectively, which were consistent with the calculated masses of 110605.18 Da, 127867.23 Da and 122605.91 Da, respectively. Therefore, the modified fusion proteases in Table 4 were not significantly truncated or degraded, as the predominantly detected masses corresponded to the calculated masses of the full-length fusion proteases.

Table 4. pET22b plasmid construct encoding NH2-des182HRV14 3C-XaaProDAP(Q241E, G242T)-COOH fusion enzyme

Example 3B

Design Full Length Dual Function NH2-XaaProDAP-HRV14-COOH protease

Using the general design, cloning and expression procedures described in Examples 1 and 3A, we also evaluated whether it was possible to obtain a functional soluble fusion protease comprising HRV14 3C at the C-terminus. The expression of 3 fusion proteases comprising the C-terminal HRV14 3C domain and the N-terminal XaaProDAP (Q241E, G242T) using 3 different N-terminal tags (His6, RL9, Trx) as described previously was evaluated. All three constructs with the HRV14 3C domain at the C-terminus were expressed as insoluble proteins as uninduced, induced, soluble and insoluble fractions as determined by SDS-PAGE (detailed data not shown). This confirms that fusion protease variants comprising HRV14 3C protease at the N-terminus and Lactococcus lactis XaaProDAP at the C-terminus unexpectedly have more optimized folding kinetics, resulting in a soluble, stable fusion protease that is easier to produce And does not require any cost prohibitive folding steps. In summary, some specifications of the protein design allowed the production of a complete fusion protease comprising the HRV14 3C and XaaProDAP proteases.

Example 4

Amplified expression and purification NH2-His- des182HRV14-LLXaaProDAP (Q241E,G242T)-COOH ( protein 20986)

To prepare larger quantities of the full-length fusion protease, amplified protease 20986 was used for further activity testing.

In 50 ml Terrific Broth medium containing 50 mg/L carbenicillin and 0.5% glucose, by shaking (Multitron Standard shaker, 50 mm amplitude, Infors HT) at 100 rpm at 37 °C, the pET22b plasmid containing protease 20986 BL21(DE3) transformants (from glycerol stocks) were propagated overnight. The next day, 7.5 ml of the overnight culture was used to inoculate 750 ml of TB medium containing 50 mg/L carbenicillin in a 2L shaker flask, and the culture was subsequently incubated at 37°C at 100 rpm. When the OD600 reached about 1.5, the culture was cooled to 20 °C for 30 min before adding 0.3 mM IPTG to induce protein. Induction was performed overnight at 20 °C, 100 rpm and cells were harvested by centrifugation at 4000 x g for 10 min. Freeze the pelleted cells for later use.

purification His-des182HRV14-LLXaaProDAP (Q241E,G242T) ( protein 20986)

To obtain a purified bifunctional fusion protease for further analysis, protease 20986 was purified by performing two consecutive purification steps.

Resuspend 14.7 g of cell pellet in 100 ml lysis buffer containing 50 mM sodium phosphate pH 7.5 and 3 µL benzonase. Cells were disrupted for one cycle at 1.4 kBar in a cell homogenizer and the cell debris was pelleted by centrifugation at 18.000 g for 20 minutes. The supernatant was then sterile filtered (0.45 microns). Purification of Protease 20986 was accomplished using AKTAExpress (GE Healthcare) in two consecutive purification steps. In the capture step, the 2X1 ml HisTrap crude column (GE Healthcare) was purified from a 100 ml sample application with a flow rate of 0.8 ml/min and the following buffers were used:

Buffer A: 50 mM Sodium Phosphate, 300 mM NaCl, 10 mM Imidazole pH 7.5

Buffer B: 50 mM Sodium Phosphate, 300 mM NaCl, 300 mM Imidazole pH 7.5

Buffer C: 50 mM Sodium Phosphate, 300 mM NaCl, 30 mM Imidazole pH 7.5

First, equilibrate the column with 10 column volumes of buffer. After application, unbound protein was removed by washing with 7 column volumes of buffer C. Use 5 column volumes of 0-100% buffer B to elute protease 20986, store the collected peaks in a ring and apply to 120 ml HiLoad S200 16/600 (GE-Healthcare) gel filtration column. Size separation was performed with a flow rate of 1.2 ml/min using 1X PBS buffer (phosphate-buffered saline, pH 7.4, composition: 8.05 mM Na2HPO4x2H2O, 1,96 mM KH2PO4, 140 mM NaCl, pH 7.4). The collected major peak fractions were analyzed by SDS-PAGE and a clear band of expected size around 100 kDa was observed. Pool the fractions containing the highest amount of protease using UV280 The measurements (NanoDrop, ThermoScientific) measured a concentration of 1,6 mg/ml. Purity was estimated to be greater than 90%, as judged by SDS-PAGE (Figure 1) and HPLC analysis.

Example 5

Plasmid constructs and expression of model fusion proteins containing basic tags

To test whether a bifunctional fusion protease could be used to remove N-terminal tags, three different model fusion proteins were prepared as protein substrates. A basic tag comprising ribosomal protein L27 from Thermotoga maritima ( T. maritima ) described in WO2008/043847 was used as a fusion partner with the following sequence: MAHKKSGGVAKNGRDSLPKYLGVKVGDGQIVKAGNILVRQRGTRFYPGKNVGMGRDFTLFALKDGRVKFETKNNKKYVSVYEE (SEQ ID NO: 16). The fusion protein was designed such that the RL27 fusion partner could be removed by the HRV14 3C enzyme and the remaining GP sequence could be removed by XaaProDAP.

The basic tag is connected to the model peptide sequence using a flexible linker containing the HRV14 cleavage site, which has the sequence SSSGGSEVLFQGP (SEQ ID NO: 17). The model peptide sequences used were human peptide YY 3-36 (PYY(3-36)), glucagon and glucagon-like peptide 1 (7-37,K34R) (GLP-1(7-37,K34R)), each having the following sequence:

PYY (3-36): IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 18)

Glucagon: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO: 19)

GLP-1(7-37, K34R): HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 20).

E. coli expression plasmids (pET22b, Novagen) were prepared encoding the three fusion proteins detailed in Table 5.

Table 5. Model fusion proteins encoded by plasmid constructs using the pET22b vector

A codon-optimized E. coli gene fragment spanning the entire fusion protein was generated by gene synthesis and ligated into the cloning site of the pET22b vector using standard cloning techniques (obtained from GenScript).

Expression model fusion protein

Expression of RL27_EVLFQGP_PYY(3-36) was performed essentially as described in Example 4 for protease 20986. Briefly, RL27_EVLFQGP_glucagon and RL27_EVLFQGP_GLP-1(7-37,K34R) were expressed as follows: E. coli BL21(DE3) was transformed with the material, plated onto LB agar plates containing 100 mg/L ampicillin, The overnight culture was dissolved in 10 ml LB culture and used to inoculate 750 ml LB containing 50 mg/ml carbenicillin in shake flasks. The shaker flask was incubated at 37°C at 100 rpm. When OD600 reached 0.4, protein expression was induced by adding 0.3 mM IPTG, and cells were harvested by centrifugation after incubation at 37°C for 3 hours.

Purification of model fusion proteins

Briefly, fusion proteins were captured by cation exchange chromatography from supernatants obtained from cell disruption essentially as described (WO2008/043847) using SP FF HiTrap 5 ml (GE Healthcare) columns on AKTA Express , the flow rate is 4 ml/min, and the buffer is as follows:

Buffer A: 50mM sodium phosphate, pH 7.0

Buffer B: 50mM sodium phosphate, 1000mM NaCl, pH 7.0.

Briefly, after the loading and washing steps, the fusion protein was eluted from the column using Buffer B. To increase the purity of the capture described below, the protein was purified by gel filtration essentially as described in Example 4, except that a S75 16/600 separation column (GE-Healthcare) was used. Purified protein was evaluated by SDS-PAGE analysis and correct intact mass was verified by LC-MS. UV280 was used to determine the concentration of the fusion protein.

Example 6

using protease 20986 and as model protein substrates RL27_EVLFQGP_PYY(3-36) enzyme reaction

Adjust concentrate RL27-HRV14-PYY(3-36) to 1X PBS, pH 0.5 mg/ml concentration in 7.4. Set up the enzyme reaction in a 22 µl reaction volume, using PBS, pH 7.4 as the enzyme reaction buffer. Using the enzyme substrate molar ratio of 1:20 or 1:40 respectively, the incubation of protease 20986 and RL27-EVLFQGP-PYY(3-36) substrate was established, and the reaction was carried out at 37°C for 3 hours (as shown in Table 6) . The purified HRV14 3C protease variant described in WO2008/043847 with an N-terminal tag (ribosomal L9 from Thermotoga maritima) was also included in this experiment. This protease, called RL9-HRV14 3C, was used in the same molar ratio as Protease 20986, but only had HRV14 3C activity. RL9-HRV14 3C具有下述序列：MKVILLRDVPKIGKKGEIKEVSDGYARNYLIPRGFAKEYTEGLERAIKHEKEIEKRKKEREREESEKILKELKKRTHVVKVKAGEGGKIFGAVTAATVAEEISKTTGLKLDKRWFKLDKPIKELGEYSLEVSLPGGVKDTIKIRVEREESSSGSSGSSGSSGPNTEFALSLLRKNIMTITTSKGEFTGLGIHDRVCVIPTHAQPGDDVLVNGQKIRVKDKYKLVDPENINLELTVLTLDRNEKFRDIRGFISEDLEGVDATLVVHSNNFTNTILEVGPVTMAGLINLSSTPTNRMIRYDYATKTGQCGGVLCATGKIFGIHVGGNGRQGFSAQLKKQYFVEKQ (SEQ ID NO: 21). As a negative control, the RL27-HRV14-PYY(3-36) substrate was also incubated in protease-free reaction buffer. Enzyme reactions were terminated by adding >0.5 M AcOH prior to LC-MS analysis.

use RL27_HRV14_PYY(3-36) Proteases as Model Substrates for Fusion Proteins 20986 result

LC-MS analysis of the enzyme reaction was performed essentially as described in Example 1 except that a C18 Aquity BEH300 C4 reversed-phase 1.0 X 100 mm column with a 1.7 µm pore size was used to ensure adequate separation and resolution of the smaller peptides evaluated . According to the manufacturer's instructions, adjust the instrument for the mass range (2000-17000 Da) and resolution (>20.000) settings. The UV215 nm chromatograms and total ion count (TIC) chromatograms were evaluated in parallel to ensure agreement between the obtained MS data and the UV215 nm traces of the peptides. The experimentally determined masses shown in the Examples below refer to the most abundant masses, eg, molecular masses with the most representative isotopic distribution based on the natural abundance of the isotopes of the detected protein. Next, mass spectral data were acquired with a mass accuracy of less than 100 ppm.

The analysis of deconvoluted mass spectrometry showed that the mass of RL27_EVLFQGP_PYY(3-36) fusion protein (control without enzyme) was 14354.17 Da. This is consistent with the calculated mass of the fusion protein without the starting methionine (14354.5 Da).

The results of the different reactions are shown in Table 6.

Table 6 : Enzyme reactions using protease 20986 from example 4 and RL27_EVLFQGP_PYY(3-36) as substrate, both incubated at 37°C for 3 hours. The experimentally determined major peaks detected in the deconvoluted mass spectra of reactions 1-4 are shown.

Reaction 1 showed that complete processing of the fusion protein was obtained after enzyme treatment at an enzyme substrate molar ratio of 1:20 and incubation at 37° C. for 1 hour ( FIG. 2 ). The observed major detected mass was 4049.9 Da, which corresponds to the mass of mature PYY(3-36) (peak #1) and released tag (peak #2). No remaining fusion protein was observed, but a peak less than 10% of peak #1 intensity was observed, which corresponds to GP-PYY(3-36). Reaction 2 showed that a 1:40 enzyme substrate ratio resulted in approximately half of GP-PYY(3-36) being processed into mature PYY(3-36) (Figure 3). Reactions 3 (Fig. 4) and (Fig. 5) show that the removal of Gly-Pro by GP-PYY(3-36) observed in reactions 1 and 2 is specific for the XaaProDAP portion of protease 20986, as it only contains the HRV14 3C structure Domain RL9-HRV14 3C protease is only able to release GP-PYY(3-36).

Experiments have shown that the fully mature PYY(3-36) peptide (4050 Da) can be released by the bifunctional fusion protease, thereby realizing the idea of the present invention.

Example 7

Include alternatives from other species 3C and XaaProDAP Full-length bifunctional fusion protease design of domains

To demonstrate that other 3C proteases and XaaProDAP enzymes can be fused to obtain functional fusion proteases with the same properties as observed for protease 20986, the 3C protease sequence from human Coxsackievirus B3 (CVB3 3C) or from Streptococcus suis ( Streptococcus suis ) XaaProDAP (Streptococcus suis XaaProDAP) replaces HRV14 3C and Lactococcus lactis XaaProDAP (LLXaaProDAP) sequences and generates new fusion protease variants. Like the 3C protease sequence from human rhinovirus 14 3C, the human Coxsackievirus B3 3C protease sequence also contains a C-terminal Q, which is deleted to obtain CVB3 3C(des183) with the following sequence:

GPAFEFAVAMMKRNSSTVKTEYGEFTMLGIYDRWAVLPRHAKPGPTILMNDQEVGVLDAKELVDKDGTNLELTLLKLNRNEKFRDIRGFLAKEEEVEVNEAVLAINTSKFPNMYIPVGQVTEYGFLNLGGTPTKRMLMYNFPTRAGQCGGVLMSTGKVLGIHVGGNGHQGFSAALLKHYFNDE(SEQ ID NO: 23).

A QG site was observed at position Q212-G213 of the S. suis XaaProDAP sequence, which is close to the 3C cleavage site (Q241-G242) determined for the L. lactis sequence.引入Glu212-Thr213取代以防止任何潜在的3C切割，由此得到下述序列：MRFNQFSFIKKETSVYLQELDTLGFQLIPDASSKTNLETFVRKCHFLTANTDFALSNMIAEWDTDLLTFFQSDRELTDQIFYQVAFQLLGFVPGMDYTDVMDFVEKSNFPIVYGDIIDNLYQLLNTRTKSGNTLIDQLVSDDLIPEDNHYHFFNGKSMATFSTKNLIREVVYVETPVDTAGTGQTDIVKLSILRPHFDGKIPAVITNSPYHETVNDVASDKALHKMEGELAEKQVGTIQVKQASITKLDLDQRNLPVSPATEKLGHITSYSLNDYFLARGFASLHVSGVGTLGSTGYMTSGDYQQVEGYKAVIDWLNGRTKAYTDHTRSLEVKADWANGKVATTGLSYLGTMSNALATTGVDGLEVIIAEAGISSWYDYYRENGLVTSPGGYPGEDLDSLTALTYSKSLQAGDFLRNKAAYEKGLAAERAALDRTSGDYNQYWHDRNYLLHADRVKCEVVFTHGSQDWNVKPIHVWNMFHALPSHIKKHLFFHNGAHVYMNNWQSIDFRESMNALLSQKLLGYENNYQLPTVIWQDNSGEQTWTTLDTFGGENETVLPLGTGSQTVANQYTQEDFERYGKSYSAFHQDLYAGKANQISIELPVTEGLLLNGQVTLKLRVASSVAKGLLSAQLLDKGNKKRLAPIPAPKARLSLDNGRYHAQENLVELPYVEMPQRLVTKGFMNLQNRTDLMTVEEVVPGQWMNLTWKLQPTIYQLKKGDVLELILYTTDFECTVRDNSQWQIHLDLSQSQLILPH (SEQ ID NO: 24).

Three new fusion protease variants were designed, comprising new 3C and XaaProDAP orthologs, and using the same His6 fusion partner (SEQ ID NO: 13) and the same intervening linker shown in Example 3 ( SEQ ID NO: 12). Protease 28994 comprised the L. lactis XaaProDAP sequence described for Protease 20986 in Example 3A, but with the N-terminal HRV14 3C domain replaced by the 3C domain from human Coxsackievirus B3 (CVB3 3C). Protease 28996 contains the HRV14 3C sequence described for Protease 20986 at the N-terminus and the S. suis XaaProDAP sequence at the C-terminus. Protease 28997 is a novel fusion protease in which both domains are replaced with orthologs of 3C and XaaProDAP proteases, whereby the protease contains the N-terminal CVB3 3C sequence and the C-terminal S. suis XaaProDAP sequence.

A plasmid construct using the pET22b vector backbone and containing the new fusion protease was obtained from GenScript. The sequence combinations encoding the designed fusion protease variants are shown in Table 7.

Table 7. PET22b plasmid constructs encoding fusion protease variants, each variant comprising N-terminal HRV14 3C or CVB3 3C and C-terminal L. lactis XaaProDAP (Q241E, G242T) or S. suis XaaProDAP (Q212E, G213T) combination

Small-scale expression and IMAC purification of the novel fusion protease constructs as described in Example 1 showed that all three novel proteases resulted in soluble intact fusion proteases comprising the novel 3C and XaaProDAP orthologs sequence.

Intact masses were determined by LC-MS analysis of IMAC purified fusion protease variants as described in Example 1, and the results confirmed the observations by SDS-PAGE. The measured masses of proteases 28994, 28996 and 28997 are 107797.8 Da, 107687.2 Da and 107964.2 Da, respectively, which are highly consistent with the calculated masses of 107798.1 Da, 107687.4 Da and 107964.8 Da, respectively. Thus, the new protease was not significantly truncated or degraded as observed for protease 20986, as the detected mass of the main peak corresponds to the calculated mass of the full-length fusion protease. Thus, all proteases 20986, 28994, 28996 and 28997 do not have self-cleavage activity capable of damaging at least one of the two proteolytically active components. In conclusion, the concept of the present invention to produce a functional 3C/XaaProDAP fusion protease was further confirmed using picornavirus 3C and other orthologs of the XaaProDAP enzymes with distinct amino acid sequences.

Example 8

Amplified expression and purification containing new 3C and XaaProDAP domain protease 28994 , 28996 and 28997

Proteases 28994, 28996 and 28997 were expressed as described in Example 4 using BL21(DE3) as the expression host. Purification was performed essentially as described in Example 4, using an IMAC step capture followed by a gel filtration step. All proteases 28994, 28996 and 28997 were successfully purified by the two-step protocol described in Example 4. Purity was estimated to be at least 90%, as judged by SDS-PAGE gel inspection and UV215 nm profile evaluation from RP separation HPLC during LC-MS analysis. MS analysis as described in Example 1 showed an estimated mass of Protease 28994 of 107797.8 Da, which was in good agreement with the expected mass (110798.1 Da, average isotopic mass). Protease 28996 had a mass of 107686.9 Da, which was in good agreement with the expected mass (107687.4 Da, average isotopic mass), and Protease 28997 had a measured mass of 107964.8 Da, which was in good agreement with the expected mass (107964.8, average isotopic mass). UV280 absorbance measurements were performed to determine fusion protein concentration (NanoDrop).

Example 9

using protease 20986 , 28994 , 28996 and 28997 enzyme reaction

Set up the enzyme reaction in a 30 µl reaction volume, using 1 X PBS, pH 7.4 as the enzyme reaction buffer. Model protein substrates for evaluating cleavage specificity include fusion proteins that, when properly processed by the enzyme, should yield human PYY(3-36) (SEQ ID NO: 18), wild-type glucagon (SEQ ID NO: 19) and GLP-1 (7-37, K34R) (SEQ ID NO: 20). The concentration of the model protein substrate was adjusted to 0.5 mg/ml using 1XPBS, pH 7.4, as described in Example 6. Changes in reaction conditions were evaluated in terms of enzyme substrate ratio as well as enzyme reaction duration and temperature. Controls containing no enzyme (1x PBS pH 7,4) or RL9-HRV14 3C (SEQ ID NO. 21 ) were included. The reaction was terminated by adding >0.5 M AcOH at the end of the experiment. Using the conditions and general settings described in Example 6, LC-MS analysis of the enzymatic reactions was performed.

as model protein substrates RL27_EVLFQGP_PYY(3-36)

Using the enzyme substrate molar ratio of 1:20 or 1:100 respectively, the incubation of protease 28994, 28996 and 28997 with the RL27-EVLFQGP-PYY (3-36) substrate was established, and the reaction was carried out at 37°C for 3 hours (as shown in Table 8). Intact mass analysis by LC-MS revealed that proteases 28994, 28996 and 28997 were able to completely process RL27_EVLFQGP_PYY(3-36) into mature PYY after incubation at 37°C for 3 hours when using an enzyme-substrate molar ratio of 1:20 (3-36) (SEQ ID NO: 18) (as observed for 20986 in Example 6). At an enzyme-substrate ratio of 1:100, lesser amounts of PYY(3-36) and GP-PYY(3-36) were detected (reactions 6, 8 and 10), and the reactions were not always complete because the detection to the complete fusion protein. At a ratio of 1:100, proteases 28996 and 28997 provided the most efficient cleavage with the lowest amount of remaining GP-PYY(3-36), with relative intensities of about 25% or About 50%. Contains RL9-HRV14 3C (SEQ ID The control of NO.21) only got GP_PYY(3-36) peak, showing that the XaaProDAP domain is responsible for completing the reaction to generate the natural N-terminus of PYY(3-36), and only unprocessed fusion protein was generated without adding enzyme. Experiments showed that combining different fusion protease variants of 3C protease from human rhinovirus or human Coxsackievirus with XaaProDAP from L. lactis or S. suis could be successfully used to process RL27_EVLFQGP_PYY(3-36) into mature PYY( 3-36), wherein Ile is the correct N-terminal amino acid residue.

Table 8 : Enzyme reactions using proteases 28994, 28996 and 28997 from Example 8 and L27_EVLFQGP_PYY(3-36) as substrate, both incubated at 37°C for 3 hours. The experimentally determined major peaks detected in the deconvoluted mass spectra of reactions 5-10 are shown.

as model protein substrates RL27_EVLFQGP_ Glucagon

Incubations of proteases 20986, 28994, 28996 and 28997 with RL27-EVLFQGP-glucagon substrate were established as described above. Intact mass analysis by LC-MS revealed that proteases 20986, 28994, 28996 and 28997 were all able to process RL27_EVLFQGP_glucagon into mature glucagon, where differences were observed in overall efficiency and specificity, using 1:100 Or 1:500 enzyme substrate ratio and use 4°C or 37°C incubation temperature (Figure 6-9). For proteases 20986, 28996, an enzyme substrate ratio of 1:500 and an incubation temperature of 4°C (Table 9, Reactions 11 and 16)) provided optimized cleavage conditions in which the fusion protein was fully processed without significant non-specificity Cut (Figures 6 and 8). The measured mass of released glucagon was consistent with the calculated mass of human wild-type glucagon of 3482.8 Da (peak #1). Proteases 28994 and 28997 were less efficient, the entire fusion protein was not fully processed (for Protease 28994) under the conditions tested, and low intensity peaks (peaks #3 and #4) indicated very limited non-specific cleavage (Table 9, Reaction 13 (Figure 7) 14 and 17 (Figure 9)). A control containing RL9-HRV14 3C (SEQ ID NO.21) produced only GP_glucagon (reaction 18, Figure 10), showing that the XaaProDAP domain is responsible for completing the reaction to produce glucagon (SEQ ID NO: 19) The natural N-terminal histidine. No added enzyme yielded only the unprocessed fusion protein, with a measured mass consistent with the calculated mass of 13787.1 Da for RL27_EVLFQGP_Glucagon without the starting methionine. This suggests that combining different fusion protease variants of picornavirus 3C protease from human rhinovirus or human coxsackievirus with XaaProDAP from L. lactis or S. suis can be successfully optimized to convert RL27_EVLFQGP_glucagon Processed to mature glucagon, where His is the correct N-terminal amino acid residue, with little or no fusion protein-related impurities.

Table 9. Enzyme reactions using proteases 20986, 28994, 28996 and 28997 and RL27_EVLFQGP_glucagon as substrate, incubated overnight at 4°C. The experimentally determined major peaks detected in the deconvoluted mass spectra of reactions 11-18 are shown.

as model protein substrates RL27_EVLFQGP_GLP-1 (7-37, K34R)

Incubation of proteases 20986, 28994, 28996 and 28997 with RL27_EVLFQGP_GLP-1 (7-37, K34R) substrate was established as described above. Intact mass analysis by LC-MS showed that proteases 20986, 28994, 28996 and 28997 were all able to fully process RL27_EVLFQGP_GLP-1 into mature GLP-1(7-37,K34R), with a determined molecular weight corresponding to a calculated mass of 3382,7 Da (Table 10, Figures 11-14). Minor differences were observed in overall efficiency and specificity, using enzyme substrate ratios of 1:100 or 1:500 and incubation temperatures of 4°C or 37°C. The observed non-specific fragment was mainly GLP-1(9-37, K34R) (calculated mass 3174,6 Da), from which the extra dipeptide was removed from the GLP-1 sequence. In this experimental setup, optimal cleavage conditions were obtained at 4°C, where the fusion protein was fully processed with very limited or no non-specific cleavage. Protease 28994 was less efficient (reactions 21 (Figure 12) and 22, Table 10) as residual fusion protein was observed after incubation. Protease 28996 provided complete processing of the fusion protein and released mature GLP-1(7-37, K34R), where no non-specific cleavage was observed with 37°C for 3h (not shown).

The most efficient reaction was obtained from Protease 20986 with optimized cleavage conditions using an enzyme substrate ratio of 1:500 and overnight incubation at 4°C with no detectable distribution of fragments from non-specific or incomplete processing (Reaction 20, Figure 11) . Similar results were obtained with proteases 28996 and 28997 (reactions 23 (Figure 13) & 25 (Figure 14)), which almost exclusively produced fully processed mature GLP-1(7-37, K34R), using a 1:100 ratio of enzyme Substrate ratio and overnight incubation at 4°C, while a small but detectable amount of unprocessed GP-GLP-1(7-37,K34R) (mature ~10% of the peak intensity) (reactions 24 & 26)). Contains RL9-HRV14 3C (SEQ ID NO:21) control produced only the expected GP_GLP-1(7-37,K34R) (reaction 27, Figure 15), showing that the XaaProDAP enzyme domain of the fusion protease is responsible for supplying the native N-terminal histidine in GLP-1 (7-37, K34R). No addition of enzyme yielded only the unprocessed fusion protein, with a measured and calculated mass of 13688.1 Da, corresponding to RL27_EVLFQGP_GLP-1 (7-37, K34R) without the starting methionine. Therefore, different fusion protease variants combining picornavirus 3C protease from human rhinovirus or human coxsackievirus with XaaProDAP from L. lactis or S. suis can be optimized to convert RL27_EVLFQGP_GLP-1(7-37, K34R) into mature GLP-1 (7-37, K34R) (SEQ ID NO:20) with His as the correct N-terminus and with no or very limited fusion protein-associated impurities.

Table 10. Enzyme reactions using proteases 20986, 28994, 28996 and 28997 and RL27_EVLFQGP_GLP-1 (7-37, K34R) as substrate, incubated overnight at 4°C. The experimentally determined major peaks detected in the deconvoluted mass spectra of reactions 19-27 are shown.

While certain features of the invention have been illustrated and described herein, many changes, substitutions, changes and equivalents will now occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such changes and changes as fall within the true spirit of the invention.

Claims (15)

1. A bifunctional fusion enzyme comprising the catalytic domain of picornavirus 3C protease and XaaProDAP. 2. the bifunctional fusion protease of claim 1, it comprises the albumen of following formula: X-Y-Z (I) or Z-Y-X (II) in X is a picornavirus 3C protease or a functional variant thereof; Y is an optional linker; Z is Xaa-Pro-dipeptidyl aminopeptidase (XaaProDAP) or a functional variant thereof; Wherein said fusion protease has substantially no self-cleavage activity capable of impairing at least one of the two proteolytic activities. 3. The bifunctional fusion protease according to any one of claims 1-2, which has formula (I), that is, the picornavirus 3C protease or its functional variant is located at the N-terminal part of the bifunctional fusion protease. 4. The bifunctional fusion protease of any one of claims 1-3, wherein X is human rhinovirus 3C protease or a functional variant thereof. 5. The bifunctional fusion protease of any one of claims 1-4, wherein X comprises SEQ ID NO: 2 or a functional variant thereof. 6. The bifunctional fusion protease of any one of claims 1-5, wherein Z is an E.C. 3.4.14.11 enzyme or a functional variant thereof. 7. The bifunctional fusion protease of claim 6, wherein Z is an enzyme from lactic acid bacteria or a functional variant thereof. 8. The bifunctional fusion protease of any one of claims 1-7, wherein Z is SEQ ID NO: 1 or a functional variant thereof. 9. The bifunctional fusion protease of any one of claims 1-6, wherein Z is an enzyme from Streptococcus or a functional variant thereof. 10. The bifunctional fusion protease of claim 10, wherein Z is SEQ ID NO: 24 or a functional variant thereof. 11. The bifunctional fusion protease of any one of claims 2-10, wherein the functional variant comprises: 1-15 amino acid substitutions, deletions or Add to. 12. The bifunctional fusion protease of any one of claims 1-11, comprising a linker Y. 13. The bifunctional fusion protease of any one of claims 1-12, comprising a tag protein linked to the N-terminus. 14. A method for preparing the bifunctional fusion protease according to any one of claims 1-13, comprising recombinantly expressing a protein comprising the bifunctional fusion protease in a host cell and subsequently isolating the bifunctional fusion protease. 15. Use of the bifunctional fusion protease of any one of claims 1-13 for the removal of N-terminal peptides or proteins from larger peptides or proteins.